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.     

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.     

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.     

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.     

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.     

An activin-A/C chimera exhibits activin and myostatin antagonistic properties

Activins are involved in many physiological and pathological processes and, like other members of the transforming growth factor-beta superfamily, signal via type II and I receptor serine kinases. Ligand residues involved in type II receptor binding are located in the two anti-parallel beta strands of the TGF-beta proteins, also known as the fingers. Activin-A mutants able to bind ActRII but unable to bind the activin type I receptor ALK4 define ligand residues involved in ALK4 binding and could potentially act as antagonists. Therefore, a series of FLAG-tagged activin-A/C chimeras were constructed, in each of which eight residues in the wrist loop and helix region (A/C 46-53, 54-61, 62-69, and 70-78) were replaced. Additionally, a chimera was generated in which the entire wrist region (A/C 46-78) was changed from activin-A to activin-C. The chimeras were assessed for ActRII binding, activin bioactivity, as well as antagonistic properties. All five chimeras retained high affinity for mouse ActRII. Of these, only A/C 46-78 was devoid of significant activin bioactivity in an A3 Lux reporter assay in 293T cells at concentrations up to 40 nM. A/C 46-53, 54-61, 62-69, and 70-78 showed activity comparable with wild type activin-A. When tested for the ability to antagonize ligands that signal via activin type II receptors, such as activin-A and myostatin, only the A/C 46-78 chimera showed antagonism (IC(50), 1-10 nM). Additionally, A/C 46-78 decreased follicle-stimulating hormone release from the LbetaT2 cell line and rat anterior pituitary cells in primary culture in a concentration-dependent manner. These data indicate that activin residues in the wrist are involved in ALK4-mediated signaling. The activin antagonist A/C 46-78 may be useful for the study and modulation of activin-dependent processes.     

Activin receptors are expressed on human lung fibroblast and activin A facilitates fibroblast-mediated collagen gel contraction

Activin A is a member of the transforming growth factor-beta superfamily that exerts its diverse biological effects through bindings to activin specific transmembrane serine/threonine kinase receptors. The fibroblast-mediated contraction of a collagen gel is thought to be a model of part of the wound-repair response and tissue contraction. In this study, we found the expression of activin type I receptors (ActR-I and ActR-IB) and type II receptor (ActR-II) on human fetal lung fibroblasts (HFL-1) by RT-PCR and immunocytochemistry. We also examined the effects of activin A on the HFL-1-mediated collagen gel contraction. Activin A stimulated collagen gel contraction in a dose dependent manner and its effect was abolished by an activin-binding protein, follistatin, that specifically suppresses activin A activities. This study demonstrated that ActR-I, ActR-1B and ActR-II are expressed on human fetal lung fibroblast and that activin A regulates fibroblast-mediated collagen gel contraction, suggesting that activin A might contribute to human lung fibroblast activities and structural remodeling observed in pulmonary fibrosis.     

The transforming growth factor beta type II receptor can replace the activin type II receptor in inducing mesoderm.

The type II receptors for the polypeptide growth factors transforming growth factor beta (TGF-beta) and activin belong to a new family of predicted serine/threonine protein kinases. In Xenopus embryos, the biological effects of activin and TGF-beta 1 are strikingly different; activin induces a full range of mesodermal cell types in the animal cap assay, while TGF-beta 1 has no effects, presumably because of the lack of functional TGF-beta receptors. In order to assess the biological activities of exogenously added TGF-beta 1, RNA encoding the TGF-beta type II receptor was introduced into Xenopus embryos. In animal caps from these embryos, TGF-beta 1 and activin show similar potencies for induction of mesoderm-specific mRNAs, and both elicit the same types of mesodermal tissues. In addition, the response of animal caps to TGF-beta 1, as well as to activin, is blocked by a dominant inhibitory ras mutant, p21(Asn-17)Ha-ras. These results indicate that the activin and TGF-beta type II receptors can couple to similar signalling pathways and that the biological specificities of these growth factors lie in their different ligand-binding domains and in different competences of the responding cells.     

Responsiveness to transforming growth factor-beta (TGF-beta) restored by genetic complementation between cells defective in TGF-beta receptors I and II.

Selection of mutant Mv1Lu mink lung epithelial cells resistant to growth inhibition by transforming growth factor-beta (TGF-beta) has led to the isolation of cell clones with distinct alterations in type I and II TGF-beta receptors. Certain mutant clones present a decreased number or complete loss of detectable type I receptor. Other clones show a loss and/or altered electrophoretic mobility of the type II receptor, with concomitant loss of the type I receptor. Using somatic cell hybridization analysis we demonstrate the recessive nature of these mutants with respect to the wild-type phenotype and define various mutant complementation groups. Among these, hybrids between cells that express only type II receptor (R mutants) and cells that express neither receptor type (DRa mutants) rescue wild-type expression of type I receptors. Moreover, these hybrids regain full responsiveness to TGF-beta 1, as measured by inhibition of DNA synthesis as well as stimulation of fibronectin and plasminogen activator inhibitor-1 production. These results provide evidence for an interaction between TGF-beta receptor components I and II and show that, in Mv1Lu cells, expression of both receptor types is required for mediation of biological responses to TGF-beta 1.     

The role of effectors of the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in patterning of tooth development.

The gene for activin betaA is expressed in the early odontogenic mesenchyme of all murine teeth but mutant mice show a patterning defect where incisors and mandibular molars fail to develop but maxillary molars develop normally. In order to understand why maxillary molar tooth development can proceed in the absence of activin, we have explored the role of mediators of activin signalling in tooth development. Analysis of tooth development in activin receptor II and Smad2 mutants shows that a similar tooth phenotype to activin betaA mutants can be observed. In addition, we identify a novel downstream target of activin signalling, the Iroquois-related homeobox gene, Irx1, and show that its expression in activin betaA mutant embryos is lost in all tooth germs, including the maxillary molars. These results strongly suggest that other transforming growth factor beta molecules are not stimulating the activin signalling pathway in the absence of activin. This was confirmed by a non-genetic approach using exogenous soluble receptors to inhibit all activin signalling in tooth development, which reproduced the genetic phenotypes. Activin, thus, has an essential role in early development of incisor and mandibular molar teeth but this pathway is not required for development of maxillary molars.     

新城疫病毒F蛋白细胞融合活性位点中保守氨基酸基因突变分析

为了确定新城疫病毒融合蛋白(F)分子上活性位点中保守氨基酸在F蛋白的细胞融合作用,弄清F细胞融合的分子机理,采用基因定点突变法,创造一个酶切位点,用酶切反应初步筛选突变株,然后用DNA序列分析进一步确定,并于真核细胞内进行表达,Giemsa染色定性和指示基因法定量检测细胞融合功能,荧光强度分析(FACS)检测表达效率情况。结果表明,NDV F第117位苯丙氨酸(F)突变成亮氨酸(L)时对细胞融合作用没有显著影响。R112和K115同为保守序列,分别突变为G时,细胞融合活性只有原来的44%,下降了56%。细胞表面表达效率没有明显的改变。N147突变为K时,细胞融合活性明显下降,只有原来的15%,而细胞表面表达效率没有明显的改变。L154为保守序列,突变为K时,细胞融合活性消失,说明L154是一个非常关键的氨基酸,对维持F蛋白的细胞融合活性非常重要。细胞表面表达效率也有所下降(为原来的94%)。D462属于高度保守氨基酸,当突变为N时,细胞融合活性消失,但经细胞表面表达效率分析证明,此突变蛋白未表达于细胞表面,证明在细胞浆转运至细胞表面的过程中发生了问题。当突变为R和E时,细胞融合活性未发生改变,但细胞表面表达效率有所下降,分别为野毒株的63%和44%。说明NDV F分子上与HN相互作用的特异性区域中的某些保守氨基酸在细胞融合中发挥着重要作用,对F蛋白的折叠、加工、转运等,发挥着不同作用,从而影响F蛋白的细胞融合作用和/或在细胞表面的表达量。     

Structural basis for a functional antagonist in the transforming growth factor beta superfamily

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.     

The gamma subunit in chloroplast F(1)-ATPase can rotate in a unidirectional and counter-clockwise manner

Desethylamiodarone (DEA) acts as a competitive inhibitor of triiodothyronine (T3) binding to the alpha1-thyroid hormone receptor (TR alpha1) but as a non-competitive inhibitor with respect to TR beta1. To gain insight into the position of the binding site of desethylamiodarone on TR beta1 we investigated the naturally occurring mutants Y321C, R429Q, P453A, P453T and the artificial mutants L421R and E457A in the ligand binding domain of human TR beta1. The IC50 values (in microM) of DEA for P453A (50 +/- 11) and P453T (55 +/- 16) mutant TR beta1 are not different from that for the wild type TR beta1 (56 +/- 15), but the IC50 values of R429Q (32 +/- 7; P<0.001) and E457A (17 +/- 3; P<0.001) are significantly lower than of the wild type. Scatchard plots and Langmuir analyses indicate a non-competitive nature of the inhibition by DEA of T3 binding to all four mutant TR beta1s tested. Mutants P453A and P453T do not influence overall electrostatic potential, and also do not influence the affinity for DEA compared to wild type. Mutant E457A causes a change from a negatively charged amino acid to a hydrophobic amino acid, enhancing the affinity for DEA. Mutant R429Q, located in helix 11, causes an electrostatic potential change from positive to uncharged, also resulting in greater affinity for DEA. We therefore postulate that amino acids R429 and E457 are at or close to the binding site for DEA, and that DEA does not bind in the T3 binding pocket itself, in line with the non-competitive nature of the inhibition of T3 binding to TR beta1 by DEA.     

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis

Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.     

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.     

Structural distortions due to missense mutations in human formylglycine-generating enzyme leading to multiple sulfatase deficiency

The major candidate for multiple sulfatase deficiency is a defective formylglycine-generating enzyme (FGE). Though adequately produced, mutations in FGE stall the activation of sulfatases and prevent their activity. Missense mutations, viz. E130D, S155P, A177P, W179S, C218Y, R224W, N259I, P266L, A279V, C336R, R345C, A348P, R349Q and R349W associated with multiple sulfatase deficiency are yet to be computationally studied. Aforementioned mutants were initially screened through ws-SNPs&GO^3D program. Mutant R345C acquired the highest score, and hence was studied in detail. Discrete molecular dynamics explored structural distortions due to amino acid substitution. Therein, comparative analyses of wild type and mutant were carried out. Changes in structural contours were observed between wild type and mutant. Mutant had low conformational fluctuation, high atomic mobility and more compactness than wild type. Moreover, free energy landscape showed mutant to vary in terms of its conformational space as compared to wild type. Subsequently, wild type and mutant were subjected to single-model analyses. Mutant had lesser intra molecular interactions than wild type suggesting variations pertaining to its secondary structure. Furthermore, simulated thermal denaturation showed dissimilar pattern of hydrogen bond dilution. Effects of these variations were observed as changes in elements of secondary structure. Docking studies of mutant revealed less favourable binding energy towards its substrate as compared to wild type. Therefore, theoretical explanations for structural distortions of mutant R345C leading to multiple sulfatase deficiency were revealed. The protocol of the study could be useful to examine the effectiveness of pharmacological chaperones prior to experimental studies.     

N-myc down-regulates activin A

N-myc oncogene amplification is frequent in human neuroblastoma and predicts poor prognosis, but the molecular consequences have remained obscure. We report here that enhanced N-myc expression correlates with low or undetectable expression of activin A, but not other closely related members of the transforming growth factor-beta superfamily. N-myc interacts with the activin A promoter, eventually inducing down-regulation of activin A mRNA and protein. This study demonstrates for the first time N-myc-induced down-regulation of a gene implicated in signal transduction. Down-regulation of activin A could deprive neuroblastomas from a signal with growth-inhibitory activities toward the tumor and its stroma and thereby permit neuroblastoma progression.