For information on BMPR-II (BMPR2) receptors, see Wishful thinking

daf-4 encodes C. elegans TGFbeta-like type II receptor

In C. elegans, the TGFbeta-like type II receptor daf-4 is required for two distinct signaling pathways. In association with the type I receptor daf-1, it functions in the dauer pathway. In addition, daf-4 is also required for body size determination and male tail patterning, roles that do not require daf-1. In an effort to determine how two different signals are transmitted through daf-4, other potential signaling partners for DAF-4 were sought. A novel type I receptor has been cloned and characterized and it is shown to be encoded by sma-6. daf-1 and sma-6 are more closely related to each other than they are to the two Drosophila genes thick veins and saxophone. Mutations in sma-6 generate the reduced body size (Sma) and abnormal male tail (Mab) phenotypes identical to those observed in C. elegans daf-4 and sma-2, sma-3, sma-4 mutants, indicating that these genes function in a common signaling pathway. However, mutations in sma-6, sma-2, sma-3, or sma-4 do not produce constitutive dauers, which demonstrates that the unique biological functions of daf-4 are mediated by distinct type I receptors functioning in parallel pathways. It is proposed that the C. elegans model for TGFbeta-like signaling, in which distinct type I receptors determine specificity, may be a general mechanism for achieving specificity in other organisms. These findings distinguish between the manner in which signaling specificity is achieved in TGFbeta-like pathways and receptor tyrosine-kinase (RTK) pathways (Krishna, 1999).

Xenopus Type II BMP Receptors

Activins and other ligands in the TGFbeta superfamily signal through a heteromeric complex of receptors. Disruption of signaling by a truncated type II activin receptor, XActRIIB (previously called XAR1), blocks mesoderm induction and promotes neuralization in Xenopus embryos. A type I activin receptor, XALK4 has been cloned and characterized. Like truncated XActRIIB, a truncated mutant (tXALK4) blocks mesoderm formation both in vitro and in vivo; moreover, an active form of the receptor induces mesoderm in a ligand-independent manner. Unlike truncated XActRIIB, however, tXALK4 does not induce neural tissue. This difference is explained by the finding that tXALK4 does not block BMP4-mediated epidermal specification, while truncated XActRIIB inhibits all BMP4 responses in embryonic explants. The neural/ectodermal fate engendered by BMP4 is likely to require another type I receptor AlK3 (BMPR1) and another type II receptor dedicated to ectodermal fate. Thus, the type I and type II activin receptors are involved in overlapping but distinct sets of embryonic signaling events (Chang, 1997).

Classical experiments performed in the amphibian embryo established that the vertebrate nervous system arises, during gastrulation, from inductive interactions between the dorsal mesoderm and the overlying ectoderm. Ectopic expression of a truncated activin type II receptor, which interferes with the signaling pathway of several members of the TGF-beta family, induces the formation of neural tissue in Xenopus explants. Since interference with type II activin receptor signaling in these animal caps unveils a neural fate, it was proposed that the formation of neural tissue in vertebrate embryos is under inhibitory control. The current study shows that in Xenopus embryos, a truncated type II activin receptor (Delta1XAR1), capable of blocking signals by several transforming growth factor (TGF)-beta family members, can induce neural tissue, suggesting neural fate is under inhibitory control. Activin and bone morphogenetic protein 4 (BMP4) can act as neural inhibitors but only BMP4 can induce epidermis in Xenopus ectodermal cells. The pluripotent mouse embryonal carcinoma cell line P19 was used to examine whether the mechanisms of ectodermal cell fate decisions are conserved among vertebrates. A P19 cell line expressing Delta1XAR1 differentiates into neurons. In addition, BMP4 inhibits retinoic acid (RA)-induced neural differentiation of P19 cells and induces keratin expression. These results suggest that in mammals, as in amphibians, neural fate is under inhibitory control and BMP4 can alter ectodermal differentiation (Hoodless, 1997).

Xenopus blastula cells activate different mesodermal genes as a concentration-dependent response to activin, which behaves like a morphogen. To understand how cells recognize morphogen concentration, radioactively labeled activin has been bound to cells and binding has been related to the choice of gene activation. The increasing occupancy of a single receptor type can cause cells to switch gene expression. Cells sense ligand concentration by the absolute number of occupied receptors per cell (100 and 300 molecules of bound activin induce Xbra and Xgsc, respectively, i.e., 2% and 6% of the total receptors) and not by a ratio of occupied to unoccupied receptors. The long duration of occupancy explains a previously described ratchet effect. These results suggest a new concept of morphogen gradient formation and interpretation that is particularly well suited to the needs of early development (Dyson, 1998).

An unexpected result of this work is that cells sense morphogen concentration and switch gene response when a remarkably small proportion of their receptors is occupied by ligand. However, this is very understandable if cells' interpretation of a morphogen gradient is envisioned in the following way: it is supposed that cells in one region of an embryo actively secrete morphogen for a few hours, during which time the concentration increases. After this, cells discontinue emitting morphogen, and its concentration decreases. It is believed that responsive cells monitor morphogen concentration continuously and respond by a ratchet mechanism to the highest concentration that they experience within their competent life. This proposed mechanism has at least two advantages for early development: (1) cells can bind ligand and respond rapidly to the morphogen and do not therefore need to wait for it to reach equilibrium. Through the ratchet effect, cells would always respond to the highest concentration that they experience within the few hours of their competent life even when the number of occupied receptors per cell might temporarily decrease, as during cell division. The ratchet effect would operate by the very high affinity of ligand for its receptors. (2) If cells were to respond at high occupancy but still measure the absolute numbers of occupied receptors, there could be some inconsistency of response due to titration of type I receptors. Under these conditions, the ligand type II receptor complexes first formed would see a much higher concentration of type I receptors than subsequent complexes. It would then be predicted that overexpression of Type I receptors would change response to activin concentration. The results presented in this paper show that this is not the case. It is concluded that, at the very low occupancies seen, both type I and type II receptors are in such excess that the few receptors actually used for signaling do not significantly reduce the overall pool of available receptors, and so increased occupancy can be directly reflected in increased signaling. The mechanism proposed can explain an apparent paradox. On the one hand, the ligand must be limiting in order to account for the concentration-dependent responses that are observed. On the other hand, the ligand must be in excess to be able to create a concentration gradient in distant cells. If this were not the case, most if not all the ligand would be sequestered by cells nearest the source, as may happen in the case of Hedgehog in Drosophila. This paradox may be explained as follows: it has been shown that cells can respond when very low levels of ligand are bound (100-300 molecules). This means that only a small proportion of the ligand in the intercellular space needs to be bound by receptors within the time available. In this way, cells are able to generate a concentration-dependent response without significantly reducing the concentration of ligand around them and therefore without disturbing the gradient (Dyson, 1998).

Chicken Type II BMP Receptors

To examine the role of BMP signaling during limb pattern formation, chicken cDNAs encoding were isolate encoding type I (BRK-1 and BRK-2) and type II (BRK-3) receptors for bone morphogenetic proteins. BRK-2 and BRK-3, which constitute dual-affinity signaling receptor complexes for BMPs, are co-expressed in condensing precartilaginous cells, while BRK-1 is weakly expressed in the limb mesenchyme. BRK-3 is also expressed in the apical ectodermal ridge and interdigital limb mesenchyme. BRK-2 is intensely expressed in the posterior-distal region of the limb bud. During digit duplication by implanting Sonic hedgehog-producing cells, BRK-2 expression is induced anteriorly in the new digit forming region as observed for BMP-2 and BMP-7 expression in the limb bud. Dominant-negative effects on BMP signaling were obtained by overexpressing kinase domain-deficient forms of the receptors. Chondrogenesis of limb mesenchymal cells is markedly inhibited by dominant-negative BRK-2 and BRK-3, but not by BRK-1. Although the bone pattern was not disturbed by expressing individual dominant-negative BRK independently, preferential distal and posterior limb truncations resulted from co-expressing the dominant-negative forms of BRK-2 and BRK-3 in the whole limb bud, thus providing evidence that BMPs are essential morphogenetic signals for limb bone patterning (Kawakami, 1996).

Mammalian Type II BMP Receptors

BRK-3 is a vertebrate type II receptor for BMP-4, distantly related to invertebrate type II receptors for BMP-2/BMP-4/dpp, such as daf-4 and punt. BRK-3 has a long carboxy-terminal sequence following intracellular kinase domain and is capable of forming a high-affinity complex with a type I receptor, BRK-2. To examine the role of BRK-2 + BRK-3 receptor complex in BMP signaling during early embryogenesis, the dominant-negative form of BRK-3 was ectopically expressed in Xenopus embryos. A secondary body axis expressing the Sonic hedgehog and N-CAM genes was induced by injecting an mRNA encoding truncated form of BRK-3 into the ventral marginal region, implicating the BMP signaling in axial mesoderm induction. Formation of the secondary axis depends on whether the deletion extends into the kinase domain, not into the carboxy-terminal tail, suggesting that the kinase domain, but not the tail region, is essential for BMP signaling (Ishikawa, 1995).

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-beta-related ligands and are known to bind to type I and type II receptor serine/threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. A cDNA has been isolated encoding a transmembrane serine/threonine kinase from human skin fibroblasts, a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhances affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. A subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection of only BRK-2 + BRK-3; this suggests that the different heteromeric BMP receptor complexes have different signaling potentials (Nohno, 1995).

Growth/differentiation factor-5 (GDF-5) is a member of the bone morphogenetic protein (BMP) family, which plays an important role in bone development in vivo. Mutations in the GDF-5 gene result in brachypodism in mice and Hunter-Thompson type chondrodysplasia in human. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors: type I and type II. However, binding abilities appear to be different among the members of the BMP family. BMP-4 binds to two different type I receptors, BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB), and a type II receptor, BMP receptor type II (BMPR-II). In addition to these receptors, osteogenic protein-1 (OP-1, also known as BMP-7) binds to activin type I receptor (ActR-I) as well as activin type II receptors (ActR-II and ActR-IIB). The binding and signaling properties of GDF-5 through type I and type II receptors has also been studied. GDF-5 induces alkaline phosphatase activity in a rat osteoprogenitor-like cell line, ROB-C26. 125I-GDF-5 binds to BMPR-IB and BMPR-II but not to BMPR-IA in ROB-C26 cells and other nontransfected cell lines. Analysis using COS-1 cells transfected with the receptor cDNAs reveals that GDF-5 binds to BMPR-IB but not to the other type I receptors when expressed alone. When COS-1 cells are transfected with type II receptor cDNAs, GDF-5 binds to ActR-II, ActR-IIB, and BMPR-II but not to transforming growth factor-beta type II receptor. In the presence of type II receptors, GDF-5 binds to different sets of type I receptors, but the binding is most efficient to BMPR-IB, as compared with the other type I receptors. A transcriptional activation signal is efficiently transduced by BMPR-IB in the presence of either BMPR-II or ActR-II after stimulation by GDF-5. These results suggest that BMPR-IB mediates certain signals for GDF-5 after forming the heteromeric complex with BMPR-II or ActR-II (Nishitoh, 1996).

Bone morphogenetic protein-2 (BMP-2) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals. BMP-2 is a homodimeric cysteine knot protein that, as a member of the transforming growth factor-ß superfamily, signals by oligomerizing type I and type II receptor serine-kinases in the cell membrane. The binding epitopes of BMP-2 for BMPR-IA (type I) and BMPR-II or ActR-II (type II) were characterized using BMP-2 mutant proteins for analysis of interactions with receptor ectodomains. A large epitope 1 for high-affinity BMPR-IA binding was detected spanning the interface of the BMP-2 dimer. A smaller epitope 2 for the low-affinity binding of BMPR-II was found to be assembled by determinants of a single monomer. Symmetry-related pairs of the two juxtaposed epitopes occur near the BMP-2 poles. Mutations in both epitopes yield variants with reduced biological activity in C2C12 cells; however, only epitope 2 variants behave as antagonists, partially or completely inhibiting BMP-2 activity. These findings provide a framework for the molecular description of receptor recognition and activation in the BMP/TGF-ß superfamily (Kirsch, 2000).

Activins and bone morphogenetic proteins (BMPs) elicit diverse biological responses by signaling through two pairs of structurally related type I and type II receptors. The crystal structure is reported of BMP7 in complex with the extracellular domain (ECD) of the activin type II receptor. The structure produces a compelling four-receptor model, revealing that the types I and II receptor ECDs make no direct contacts. Nevertheless, truncated receptors lacking their cytoplasmic domain retain the ability to cooperatively assemble in the cell membrane. Also, the affinity of BMP7 for its low-affinity type I receptor ECD increases 5-fold in the presence of its type II receptor ECD. Taken together, these results provide a view of the ligand-mediated cooperative assembly of BMP and activin receptors that does not rely on receptor-receptor contacts (Greenwald, 2003).

Characterization of the kinase activity of type II TGF-beta receptors

The type II receptors of TGFß and activin recognize these ligands free in the medium, whereas their type I receptors do not. The type I receptors recognize ligand-bound type II receptors, forming an oligomeric complex, probably a heterotetramer. The BMP receptor system is somewhat different. In this case, the type II receptors and the type I receptors both separately have low affinity for the ligand and together achieve high affinity binding. A central event in the generation of signals by type I and type II complexes is phosphorylation of the type I receptor. This is likely to be catalyzed by the type II receptor kinase. The activity of this kinase is required for phosphorylation in the cell, and it phosphorylates recominant type I receptor in vitro. Phosphorylation occurs in a cluster of five serine and threonine residues in the GS domain, a highly conserved region next to the N-terminus of the kinase domain in all type I receptors. The type II receptors have kinase activity that does not seem to be augmented by ligand binding. The ligand may be acting as an adaptor that brings a substrate-the type I receptor-to the primary receptor kinase (Massagué, 1996).

The type I and type II receptors for transforming growth factor-beta (TGF-beta) are structurally related transmembrane serine/threonine kinases, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-beta signaling, the kinase activity of the type II TGF-beta receptor was characterized. The type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate occurs not only on serine and threonine but also on tyrosine. The kinase activity of the cytoplasmic domain is inhibited by the tyrosine kinase inhibitor tyrphostin. Sites of tyrosine phosphorylation are localized to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibits the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-beta receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-beta receptor signaling (Lawler, 1997).

Transforming growth factor-beta (TGFbeta) signaling requires phosphorylation of the type I receptor TbetaR-I by TbetaR-II. Although TGFbeta promotes the association of TbetaR-I with TbetaR-II, these receptor components have affinity for each other that can lead to their ligand-independent activation. The immunophilin FKBP12 (a cytosolic protein known to bind the immunosuppressants FK506 and rapamycin) binds to TbetaR-I and inhibits its signaling function. FKBP12 binding to TbetaR-I involves the rapamycin/Leu-Pro binding pocket of FKBP12 and a Leu-Pro sequence located next to the activating phosphorylation sites in TbetaR-I. Mutations in the binding sites of FKBP12 or TbetaR-I abolish the interaction between these proteins, leading to receptor activation in the absence of added ligand. FKBP12 does not inhibit TbetaR-I association with TbetaR-II; rather, it inhibits TbetaR-I phosphorylation by TbetaR-II. Rapamycin, which blocks FKBP12 binding to TbetaR-I, reverses the inhibitory effect of FKBP12 on TbetaR-I phosphorylation. By impeding the activation of TGFbeta receptor complexes formed in the absence of ligand, FKBP12 may provide a safeguard against leaky signaling resulting from the innate tendency of TbetaR-I and TbetaR-II to interact with each other (Chen, 1997).

The type II transforming growth factor-beta (TGF-beta) receptor Ser/Thr kinase (TbetaRII) is responsible for the initiation of multiple TGF-beta signaling pathways; loss of its function is associated with many types of human cancer. TbetaRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane-proximal segment outside the kinase domain, undergoes intra-molecular autophosphorylation, which is essential for the activation of TbetaRII kinase activity, activation of TbetaRI and TGF-beta-induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T-loop region in a three-dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an intermolecular mechanism. Phosphorylation of Ser409 is essential for TbetaRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild-type to induce TbetaRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, these results show that TbetaRII phosphorylation is regulated intricately and affects TGF-beta receptor signal transduction both positively and negatively (Luo, 1997).

Mutation of type II TGF-beta and activin receptors

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, transforming growth factor-beta (TGF-beta) signaling has been functionally inactivated in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocks signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice show increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities are not observed in the liver. Additional abnormalities in the pancreas include fibrosis, neoangiogenesis and mild macrophage infiltration; these are associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments (Bottinger, 1997).

Members of the TGF-beta superfamily are important regulators of skeletal development. TGF-betas signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-betas in the development and maintenance of the skeleton, transgenic mice (MT-DNIIR-4 and -27) were generated that express the DNIIR in skeletal tissue. DNIIR mRNA expression is localized to the periosteum/perichondrium, synovium, and articular cartilage. Lower levels of DNIIR mRNA are detected in growth plate cartilage. Transgenic mice frequently show bifurcation of the xiphoid process and sternum. They also develop progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembles human osteo-arthritis. The articular surface is replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium is hyperplastic, and cartilaginous metaplasia is observed in the joint space. The hypothesis was tested that TGF-beta is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen is increased in the growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining is detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrate increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/perichondrium to inhibit cartilage differentiation. The data suggest that TGF-beta may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-beta promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans (Serra, 1997).

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, transgenic mice were generated that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibit a thickened and wrinkled skin; histologically the epidermis is markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd shows a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminishes, and by 10-14 days after birth the transgenic mice are indistinguishable from their normal siblings. However, when F1 mice are mated to homozygosity, perinatal lethality occurs due to the severe hyperkeratotic phenotype, which restricts movement. Cultured primary keratinocytes from delta beta RII mice also exhibit an increased rate of growth in comparison with nontransgenic controls, and are resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis (Wang, 1997).

Disruption of the type IIB activin receptor (ActRIIB) by gene targeting results in altered expression of multiple Hox genes and abnormal patterning of the vertebrae, similar to but more severe than retinoic acid (RA)-induced anterior transformation. RA and ActRIIB mutation have synergistic effects on vertebral patterning. Activin, Vg-1 and type II activin receptors have all been implicated in regulation of lateral asymmetry during chick and Xenopus development. The ActRIIB-/- mice die after birth with complicated cardiac defects including randomized heart position, malposition of the great arteries, and ventricular and atrial septal defects. In addition, the heart anomalies are associated with right pulmonary isomerism and splenic abnormalities, recapitulating the clinical symptoms of the human asplenia syndrome. These findings provide genetic evidence that the ActRIIB-mediated signaling pathway plays a critical role in patterning both anteroposterior and left-right axes in vertebrate animals (Oh, 1997).

Overexpression of a truncated form of an activin receptor Type IIB abolishes activin responsiveness and mesoderm formation in vivo. The extracellular domains of Xenopus Type IIA activin receptor XSTK9 and Type IIB receptor differ from one another by 43%. A comparison of their intracellular domains shows that they differ by 25%. The two receptors were also compared to see if they have different functions. Simple overexpression of the wild-type receptors reveals minimal differences, but experiments with dominant negative mutants of each receptor show qualitatively distinct effects. While truncated (kinase domain-deleted) Type IIB receptors cause axial defects, truncated Type IIA receptors cause formation of secondary axes, similar to those seen by overexpression of truncated receptors for BMP-4, another TGFbeta family member. Furthermore, in animal cap assays, truncated Type IIB receptors inhibit induction of mesodermal markers, while truncated Type IIA receptors suppress induction only of ventral markers; the anterior/dorsal marker goosecoid is virtually unaffected. The suppression of ventral development by the Type IIA truncated receptor suggests either that the truncated Type IIA receptor interferes with ventral BMP pathways, or that activin signaling through the Type IIA receptor is necessary for ventral patterning (New, 1997).

The type II activin receptors, ActRIIA and ActRIIB, have been shown to play critical roles in axial patterning and organ development in mice. To investigate whether their function is required for mesoderm formation and gastrulation as implicated in Xenopus studies, mice were generated carrying both receptor mutations by interbreeding the ActRIIA and ActRIIB knockout mutants. Embryos homozygous for both receptor mutations were growth arrested at the egg cylinder stage and did not form mesoderm. Further analyses revealed that ActRIIA(-/-)ActRIIB(+/-) and about 15% of the ActRIIA(-/-) embryos fail to form an elongated primitive streak, resulting in severe disruption of mesoderm formation in the embryo proper. Interestingly, similar gastrulation defects are observed in ActRIIA(-/-)nodal(+/-) double mutants, which, if they develop beyond the gastrulation stage, display rostral head defects and cyclopia. These results provide genetic evidence that type II activin receptors are required for egg cylinder growth, primitive streak formation, and rostral head development in mice (Song, 1999).

Chimeric TGF-beta type II receptors

Two different chimeric TGF-beta superfamily receptors were generated: TbetaR-I/BMPR-IB, containing the extracellular domain of TGF-beta type I receptor (TbetaR-I) and the intracellular domain of bone morphogenetic protein type IB receptor (BMPR-IB), and TbetaR-II/ActR-IIB, containing the extracellular domain of TGF-beta type II receptor (TbetaR-II) and the intracellular domain of activin type IIB receptor (ActR-IIB). In the presence of TGF-beta1, TbetaR-I/BMPR-IB and TbetaR-II/ActR-IIB form heteromeric complexes with wild-type TbetaR-II and TbetaR-I, respectively, upon stable transfection in mink lung epithelial cell lines. TbetaR-II/ActR-IIB restores the responsiveness upon transfection in mutant cell lines lacking functional TbetaR-II with respect to TGF-beta-mediated activation of a transcriptional signal, extracellular matrix formation, growth inhibition, and Smad phosphorylation (see Drosophila Mothers against DPP). TbetaR-I/BMPR-IB and TbetaR-II/ActR-IIB forms a functional complex in response to TGF-beta and induced phosphorylation of Smad1. However, complex formation is not enough for signal propagation, which is shown by the inability of TbetaR-I/BMPR-IB to restore responsiveness to TGF-beta in cell lines deficient in functional TbetaR-I. The TGF-beta1-induced complex between TbetaR-II/ActR-IIB and TbetaR-I stimulates endogenous Smad2 phosphorylation, a TGF-beta-like response. This observation is in agreement with the current model for receptor activation, in which the type I receptor determines signal specificity (Persson, 1997).

Heteromerization of the Type I and Type II Activin Receptors

Activins and inhibins belong to the transforming growth factor (TGF-beta)-like superfamily and exert their effects on a broad range of cellular targets by modulating cell differentiation and proliferation. Members of this family interact with two structurally related classes of receptors (type I and type II), both containing a serine/threonine kinase domain. When expressed alone, the type II but not the type I activin receptor can bind activin. However, the presence of a type I receptor is required for signaling. For TGF-1, ligand binding to the type II receptor results in the recruitment and transphosphorylation of the type I receptor. Transient overexpression of the two types of activin receptors results in ligand-independent receptor heteromerization and activation. Nevertheless, activin addition to the transfected cells increases complex formation between the two receptors, suggesting a mechanism of action similar to that observed for the TGF-beta receptor. A stable cell line was generated, overexpressing upon induction the two types of human activin receptors, in the human erythroleukemia cell line K562. Activin specifically induces heteromer formation between the type I and type II receptors in a time-dependent manner. Activin signal transduction mediated through its type I and type II receptors results in an increase in the hemoglobin content of the cells and limits their proliferation. The inhibin antagonistic effects on activin-induced biological responses are mediated through a competition for the type II activin receptor but also require the presence of an inhibin-specific binding component (Lebrun, 1997).

TGF-beta type II receptor and tooth formation

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through their cognate receptors to determine cell phenotypes during embryogenesis. Studies on the regulation of first branchial arch morphogenesis have identified critical components of a hierarchy of different TGF-beta isoforms and their possible functions in regulating tooth and cartilage formation during mandibular morphogenesis. The hypothesis that TGF-beta type II receptor (TGF-beta IIR) is a critical component in the TGF-beta signaling pathway regulating tooth formation has been tested. To establish the precise location of TGF-beta ligand and its cognate receptor, detailed analyses were performed of the localization of both TGF-beta2 and TGF-beta IIR during initiation and subsequent morphogenesis of developing embryonic mouse tooth organs. A possible autocrine functional role for TGF-beta and its cognate receptor (TGF-beta IIR) has been inferred due to the temporal and spatial localization patterns during the early inductive stages of tooth morphogenesis. Also, loss of function of TGF-beta IIR in a mandibular explant culture model results in the acceleration of tooth formation to the cap stage while the mandibular explants in the control group only show bud stage tooth formation. In addition, there is a significant increase in odontogenic epithelial cell proliferation following TGF-beta IIR abrogation. These results demonstrate, for the first time, that abrogation of the TGF-beta IIR stimulates embryonic tooth morphogenesis in culture and reverses the negative regulation of endogenous TGF-beta signaling upon enamel organ epithelial cell proliferation (Chai, 1999).

TGF-beta type II receptor and branching morphogenesis

Although exogenous transforming growth factor-beta (TGF-beta) is known to inhibit branching morphogenesis in mouse embryonic lungs in culture, whether the principal negative function of endogenous TGF-beta signaling resides in lung epithelium or mesenchyme remains unresolved. A recombinant adenovirus was constructed, containing a mutated human TGF-beta type II receptor with a truncated cytoplasmic kinase domain. Could this dominant-negative receptor abolish epithelium-specific endogenous TGF-beta signaling? The recombinant adenovirus was introduced into lung explants via intra-tracheal micro-injection. This results in over-expression of exogenous truncated TGF-beta type II receptor only in airway epithelium, not in mesenchyme, as assessed by mRNA level and protein localization. Blockade of endogenous TGF-beta receptor signaling in epithelial endoderm by the mutated dominant-negative TGF-beta type II receptor results in significant (65%) stimulation of epithelial branching morphogenesis, while exogenous TGF-beta no longer downregulates epithelial PCNA immunoreactivity and surfactant protein C (SP-C) expression. The mitogenic responses to epidermal growth factor (EGF) and platelet-derived growth factor, PDGF-AA, are potentiated by 33% and 31%, respectively. It is concluded that epithelium-specific adenovirus-mediated over-expression of a dominant-negative TGF-beta type II receptor completely and specifically abolishes the anti-proliferative effects of both endogenous and exogenous TGF-beta. Therefore, epithelium-specific TGF-beta signaling is sufficient to negatively regulate embryonic lung-branching morphogenesis in culture. It is speculated that abrogation of TGF-beta signaling stimulates lung morphogenesis by potentiating the inductive and permissive effects of other endogenous peptide growth factors, such as EGF and PDGF-AA (Zhao, 1998).

TGF-beta type II receptors and tumorogenesis

Invasive growth of epithelial tumor cells, a major cause of death from cancer in humans, involves loss of epithelial polarity and dedifferentiation. Transforming growth factor beta (TGFbeta) is regarded as a major tumor suppressor during early tumor development because it inhibits cell-cycle progression and tumor growth. However, many dedifferentiated, late-stage tumors are resistant to growth inhibition by TGFbeta, and even secrete TGFbeta. In line with this, TGFbeta is involved in angiogenesis, wound healing and epithelial-mesenchymal transition (EMT) during development. Ha-Ras-transformed mammary epithelial cells (EpRas) undergo TGFbeta-induced EMT maintained via a TGFbeta autocrine loop. Is signal transduction by the TGFbeta receptor (TGFbetaR) required for local tumor cell invasion and metastasis? To answer this question, a dominant-negative type II TGFbetaR (TGFbetaRII-dn) was expressed using retroviral vectors in EpRas cells and highly metastatic mesenchymal mouse colon carcinoma cells (CT26). In both cell types, TGFbetaRII-dn blocks TGFbetaR signaling and heavily delays tumor formation. In EpRas cells, TGFbetaRII-dn prevents EMT. In the dedifferentiated mesenchymal CT26 cells, TGFbetaRII-dn causes mesenchymal-to-epithelial transition and inhibits their in vitro invasiveness in several assays. In addition, TGFbetaRII-dn completely abolishes metastasis formation by CT26 cells. Furthermore, several human carcinoma lines lose in vitro invasiveness when treated with neutralizing TGFbeta antibodies or soluble receptor variants. Finally, human colon carcinoma cells (hnPCC) expressing a mutated, non-functional TGFbetaRII are non-invasive in vitro, a defect restored by re-expressing wild-type TGFbetaRII. It is concluded that cell-autonomous TGFbeta signaling is required for both induction and maintenance of in vitro invasiveness and metastasis during late-stage tumorigenesis. TGFbetaRII therefore represents a potential target for therapeutical intervention in human tumorigenesis (Oft, 1998).

Signaling downstream of TGF-beta type II receptors

Smad2 and Smad3 are structurally highly similar and mediate TGF-beta signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF-beta or activin stimulation. Smad2 and Smad3 interact with the kinase-deficient TGF-beta type I receptor (TbetaR)-I after it is phosphorylated by TbetaR-II kinase. TGF-beta1 induces phosphorylation of Smad2 and Smad3 in cultured Mv1Lu mink lung epithelial cells. Smad4 is found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF-beta1 stimulation. Similar results are obtained using HSC4 cells, which are also growth-inhibited by TGF-beta. Smads 2 and 3 interact with Smad4 after TbetaR activation in transfected COS cells. In addition, TbetaR-activation-dependent interaction is observed between Smad2 and Smad3. Smads 2, 3 and 4 accumulate in the nucleus upon TGF-beta1 treatment in Mv1Lu cells, and show a synergistic effect in a transcriptional reporter assay using the TGF-beta-inducible plasminogen activator inhibitor-1 promoter. Dominant-negative Smad3 inhibits the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF-beta induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF-beta signal transduction (Nakao 1997).

punt: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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