Jeffrey L. Wrana

TGFβ signals through a heteromeric protein kinase receptor complex

Transforming growth factor beta (TGF beta) binds with high affinity to the type II receptor, a transmembrane protein with a cytoplasmic serine/threonine kinase domain. We show that the type II receptor requires both its kinase activity and association with another TGF beta-binding protein, the type I receptor, to signal growth inhibition and early gene responses. Receptors I and II associate as interdependent components of a heteromeric complex: receptor I requires receptor II to bind TGF beta, and receptor II requires receptor I to signal. This mode of operation points to fundamental differences between this receptor and the protein-tyrosine kinase cytokine receptors.

Betaglycan presents ligand to the TGFβ signaling receptor

Abstract Transforming growth factor β (TGFβ) signals through a heteromeric protein kinase receptor that has a limited ability to bind ligand. This limitation is overcome by the action of betaglycan (TGFβ type III receptor), a separate TGFβ-binding membrane protein of previously unknown function. Betaglycan presents TGFβ directly to the kinase subunit of the signaling receptor, forming a high affinity ternary complex. Membrane betaglycan increases TGFβ binding to the signaling receptor, enhances cell responsiveness to TGFβ, and eliminates marked biological differences between TGFβ isoforms. Thus, betaglycan is a direct regulator of TGFβ access to the signaling receptors.

Identification of human activin and TGFβ type I receptors that form heteromeric kinase complexes with type II receptors

Transforming growth factor beta (TGF beta) and activin each bind to pairs of membrane proteins, known as receptor types I and II, that associate to form a signaling complex. We report that TSR-I and ActR-I, two human transmembrane serine/threonine kinases distantly related to TGF beta and activin type II receptors, act as type I receptors for these factors. TSR-I is a type I receptor shared by TGF beta and activin, whereas ActR-I is an activin type I receptor. ActR-I, but not TSR-I, signals a particular transcriptional response in concert with activin type II receptors. The results indicate that type I receptors are transmembrane protein kinases that associate with type II receptors to generate diverse heteromeric serine/threonine kinase complexes of different signaling capacities.

GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex.

The TGF‐beta type II receptor (T beta R‐II) is a transmembrane serine/threonine kinase that, upon ligand binding, recruits and phosphorylates a second transmembrane kinase, T beta R‐I, as a requirement for signal transduction. T beta R‐I is phosphorylated by T beta R‐II in the GS domain, a 30 amino acid region preceding the kinase domain and conserved in type I receptors for other TGF‐beta‐related factors. The functional role of seven serines and threonines in the T beta R‐I GS domain was investigated by mutational analysis. Five of these residues are clustered (TTSGSGSG) in the middle of the GS domain. Mutation of two or more of these residues impairs phosphorylation and signaling activity. Two additional threonines are located near the canonical start of the kinase domain, and their individual mutation to valine strongly inhibits receptor phosphorylation and signaling activity. Replacement of one of these residues, Thr204, with aspartic acid yields a product that has elevated in vitro kinase activity and signals anti‐proliferative and transcriptional responses in the absence of ligand and T beta R‐II. The identification of constitutively active T beta R‐I forms confirms the hypothesis that this kinase acts as a down‐stream signaling component in the TGF‐beta receptor complex, and its activation by T beta R‐II or by mutation is necessary and sufficient for propagation of anti‐proliferative and transcriptional responses.

The TGF-β family and its composite receptors

In their search for regulators of animal growth and development, biologists have often come upon members of the transforming growth factor beta (TGF-beta) family and have realized that these are among the most versatile carriers of growth and differentiation signals. New evidence suggests that these factors signal through receptors with remarkable structures. Each receptor is a complex of two distantly related transmembrane serine/threonine kinases that are both essential for signalling. TGF-beta and related factors have at their disposal a repertoire of such receptors, a feature that could account for their multifunctional nature.

Novel activin receptors : distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors

We have cloned ActR-IIB, which encodes four new activin receptor isoforms belonging to the protein serine/threonine kinase receptor family. Two of the ActR-IIB isoforms have higher affinity for activin A than the previously cloned activin receptor and differ from each other by the inclusion of an alternatively spliced segment in the cytoplasmic juxtamembrane region. A second alternative splicing event generates two additional receptor isoforms that lack a proline cluster in the external juxtamembrane region and have lower affinity for activin A. All isoforms bind inhibin A with low affinity. Thus, the repertoire of activin receptors includes species that differ in ligand binding affinity, cytoplasmic domain structure, or both. This receptor heterogeneity might underlie the sharply different responses that activin can elicit in a dose- or cell-specific manner.

Type I receptors specify growth-inhibitory and transcriptional responses to transforming growth factor beta and activin.

Transforming growth factor beta (TGF-beta) and activin bind to receptor complexes that contain two distantly related transmembrane serine/threonine kinases known as receptor types I and II. The type II receptors determine ligand binding specificity, and each interacts with a distinct repertoire of type I receptors. Here we identify a new type I receptor for activin, ActR-IB, whose kinase domain is nearly identical to that of the recently cloned TGF-beta type I receptor, T beta R-I. ActR-IB has the structural and binding properties of a type I receptor: it binds activin only in the presence of an activin type II receptor and forms a heteromeric noncovalent complex with activin type II receptors. In Mv1Lu lung epithelial cells, ActR-IB and T beta R-I signal a common set of growth-inhibitory and transcriptional responses in association with their corresponding ligands and type II receptors. The transcriptional responses include elevated expression of fibronectin and plasminogen activator inhibitor 1. Although T beta R-I and ActR-IB are nearly identical in their kinase domains (90% amino acid sequence identity), their corresponding type II receptor kinase domains are very different from each other (42% amino acid sequence identity). Therefore, signaling of a specific set of responses by TGF-beta and activin correlates with the presence of similar type I kinases in their complex. Indeed, other TGF-beta and activin type I receptors (TSR-I and ActR-I) whose kinase domains significantly diverge from those of T beta R-I and ActR-IB do not substitute as mediators of these growth-inhibitory and extracellular matrix transcriptional responses. Hence, we conclude that the type I receptor subunits are primary specifiers of signals sent by TGF-beta and activin receptor complexes.

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs. Images

Characterization and relationship of dpp receptors encoded by the saxophone and thick veins genes in Drosophila

The dpp/BMP family of TGF beta-related factors controls numerous events in pattern formation and morphogenesis. How these polypeptide signals are received and transduced by target cells is largely unknown. We combine molecular and genetic approaches to establish that the Drosophila saxophone (sax) gene encodes a dpp receptor. We compare the structural properties and expression patterns of sax with a second dpp receptor encoded by the thick veins (tkv) gene. While the sax gene is expressed ubiquitously, tkv is expressed in a highly localized and dynamic pattern during development. Some, but not all, of the tkv expression pattern parallels that of dpp. Ubiquitous expression of a tkv transgene rescues both tkv and sax loss-of-function mutations. Thus, there is at least partial functional overlap of the sax and tkv receptors in vivo. We consider these observations in terms of possible ligand-receptor interactions during Drosophila development.

Drosophila Dpp signaling is mediated by the punt gene product: A dual ligand-binding type II receptor of the TGFβ receptor family

Signaling by TGF beta-related factors requires ligand-induced association between type I and type II transmembrane serine/threonine kinases. In Drosophila, the saxophone (sax) and thick veins (tkv) genes encode type I receptors that mediate signaling by decapentaplegic (dpp), a member of the bone morphogenetic protein (BMP) subgroup of TGF beta-type factors. In this report, we demonstrate that the Drosophila punt gene encodes atr-II, a previously described type II receptor that on its own is able to bind activin but not BMP2, a vertebrate ortholog of dpp. Mutations in punt produce phenotypes similar to those exhibited by tkv, sax, and dpp mutants. Furthermore, punt will bind BMP2 in concert with tkv or sax, forming complexes with these receptors. We suggest that punt functions as a type II receptors for dpp and propose that BMP signaling in vertebrates may also involve sharing of type II receptors by diverse ligands.